The long term goal of these studies is to define the host-virus interactions that occur during hepatitis C virus (HCV) infection with the hope that this knowledge will lead to the discovery of improved therapeutic approaches to this important human pathogen. HCV is a noncytopathic, positive-strand RNA virus that causes acute and chronic hepatitis and hepatocellular carcinoma. Approximately 2-4 million persons are chronically infected by HCV in the USA and 170 million people are chronically infected worldwide, many of whom will die from liver failure and hepatocelluar carcinoma. The current application was stimulated by a desire to understand how HCV can survive in the face of the massive type 1 interferon (IFN) stimulated gene (ISG) expression response it induces in the infected liver. The confusion in the field surrounding this important question led us to ask if HCV inhibits ISG expression at the effector level. Using a recently developed in vitro HCV infection model system, we showed that although HCV- infected cells responded to IFN? as strongly as uninfected cells at the ISG transcriptional level, translation of the same ISGs was severely attenuated in the infected cells. Importantly, the kinetics of this effect coincided with HCV-induced phosphorylation of protein kinase R (PKR) and its substrate eIF2? which must be hypophosphorylated to support capped mRNA translation. PKR- activation by viral double stranded RNA usually triggers an antiviral innate response by inhibiting viral mRNA translation. In contrast, our surprising results suggest that PKR activation appears to have a proviral effect on HCV by suppressing cellular ISG mRNA translation. The uniqueness of HCV in this regard may reflect its dependence on an eIF2?-independent IRES element for translation which others have shown to be much less sensitive to inhibition by PKR activation than other IRES elements and capped cellular messenger RNA. In Specific Aim 1 in this application, we will identify the molecular nature of the HCV-induced/related signal that activates PKR. In Specific Aim 2, we will determine if the virus targets PKR indirectly via one of its regulatory proteins (e.g. PACT, TRBP, P58). In Specific Aim 3, we will determine how PKR phosphorylation confers a survival advantage for HCV. Finally, in Specific Aim 4, we will determine if, in addition to eIF2? phosphorylation, PKR activates other pathways (e.g NFkB, p38, JNK, FADD) that could regulate either HCV infection or the interferon response. PUBLIC HEALTH RELEVANCE: Over 170 million people throughout the world are chronically infected by HCV, 2-4 million of whom live in the United States. Twenty percent of these patients will develop cirrhosis of the liver and about 5% will die from liver failure and cancer. The work described in this proposal will improve our understanding of the host-virus interactions that regulate this infection with the hope that we will identify vulnerabilities in the HCV life cycle that can be exploited to develop safe and effective antiviral drugs to alleviate the human suffering and socioeconomic burden of this worldwide threat to public health.